Sugar transport in endothelial cells of the blood brain barrier and erythrocytes is mediated by GLUT1. These cells rapidly respond to metabolic stress with accelerated glucose transport and distribution to target tissues. This proposal outlines our continuing efforts to understand the molecular mechanism of acute, metabolic control of GLUT1 and its role in organismal carbohydrate homeostasis. We show that sugar transport through GLUT1 involves two steps: 1) Very rapid, ATP-insensitive membrane translocation into a GLUT1 cytoplasmic cage; 2) Fast release from the cage into cytoplasm. Release from cage to cytosol rate-limits transport and is allosterically regulated by ATP, AMP and pH. ATP promotes a third transport event that limits sugar release to only 66% of the cell water. This is observed as fast sugar uptake into 66% of the cell water followed by slow uptake into the remaining 34%. This may result from ATP- dependent preferential transport of [unreadable]-sugar anomers. ATP causes structural changes in cytoplasmic loop 6 and the C-terminus protecting these domains from proteolytic attack, IgG binding and lysine covalent modification at residues 245, 255, 256 and 477. Site-directed mutagenesis and peptide mapping suggest that the GLUT1 ATP-binding domain includes endofacial loop 8. Regulation by ATP is blunted by GLUT1-C-terminus targeted IgGs and is mimicked by a GLUT1-C-terminal peptide (residues 473-492). This proposal critically evaluates an integrated model for GLUT1 regulation. We ask: What GLUT1 structural changes occur upon GLUT1-ATP interaction? How do these changes influence GLUT1 function? Specific Aim 1 tests the hypothesis that cytoplasmic loop 8 is the ATP binding domain by ESI MS-MS sequence analysis of chymotryptic digests of GLUT1 covalently modified with photoreactive nucleotide analogs. Mutagenesis of labeled amino acids will evaluate their roles in ATP binding and GLUT1 regulation. Specific Aim 2 tests the hypothesis that the GLUT1 C-terminus and cytoplasmic loop 6 play a primary role in transport regulation by swapping GLUT1 loop 6 and C-terminal domains with equivalent sequence from ATP- insensitive isomers (GLUT3 & 4) and testing chimeras for functional and structural ATP-sensitivity. Specific Aim 3 tests the hypothesis that transport regulation is mediated by an auto-inhibitory mechanism involving GLUT1 C-terminus interactions with Loop 6. We investigate the molecular mechanism by which a 20-residue, GLUT1 C-terminal peptide mimics ATP-regulation of sugar transport in red cell ghosts. Specific Aim 4 tests the hypothesis that ATP-induced fast and slow sugar uptake phases in RBCs result from ATP-dependent differential transport of ([unreadable]- and a-sugars. We apply four critical tests of the anomer theory and ask whether C-terminus-loop 6 interactions mediate ATP-induced GLUT1 preference for [unreadable]-sugar. [unreadable] [unreadable] [unreadable]